Lung cancer accounts for one-fourth of all cancer deaths in the U.S. Adenocarcinoma (ADC) is the most prevalent lung cancer histological subtype, accounting for 40% of cases and 65,000 deaths in the U.S. each year. Notch signaling is aberrantly activated in 40% of lung ADCs and is associated with poor overall survival. We and others have shown that Notch regulates cancer stem cells (CSCs), epithelial-mesenchymal transition (EMT), and chemotherapy resistance in lung ADC, contributing to tumor progression. Unfortunately, as with many therapies against pleiotropic pathways, targeted therapies against Notch, including gamma secretase inhibitors (GSIs), have been largely unsuccessful due to dose-limiting toxicities or resistance. Thus, identification and successful targeting of novel effectors of Notch signaling may alleviate resistance and toxicities caused by generalized Notch inhibition while providing effective therapeutics for the treatment of lung ADC. Based upon microRNA microarrays performed on human lung ADC cell lines overexpressing or repressing Notch1 and tissues from a Notch1-induced lung ADC mouse model, we propose that miR-21 may be a direct Notch1 target microRNA (miRNA). Functionally, miR-21, which is regulated by Kras and TGF, has roles in CSCs, EMT, and chemoresistance and it has been shown that overexpression of miR-21 is associated with poor prognosis in lung cancer patients. Thus, the central hypothesis of this work is that miR-21 is a direct Notch1 target miRNA that participates in mediating Notch1-induced CSC phenotypes, EMT, and resistance to chemotherapy in lung ADC. This research aims to define miR-21 as a direct target of canonical Notch1 signaling in lung ADC through mechanistic gain- and loss-of-function studies, as well as demonstration that Notch1 binds to a putative RBP-J? binding site on miR-21's promoter. Additionally, CSC and EMT functional assays will test miR-21's role in mediating Notch1's contribution to CSC maintenance and EMT induction. Since it has been shown previously that miR-21 contributes to paclitaxel resistance, this research aims to demonstrate that GSI-mediated repression of miR-21 in combination with paclitaxel contributes to synergistic anti-tumor activity in Kras-wild type lung ADC. Synergy will be tested in preclinical mouse xenograft studies, while knocking down or overexpressing miR-21 in combination with paclitaxel and/or GSIs will assess the role of miR-21 in mediating drug synergy. These proposed studies will provide new understandings of miRNA-mediated oncogenic pathways in lung ADC progression and chemoresistance, highlighting miR-21 as a potential mediator of Notch1's role in lung ADC. In the end, the results could lead to elucidation of novel targets for lung cancer therapy while bypassing the toxicities and resistance caused by targeting pleiotropic and redundant oncogenic pathways.